Figures & data
Figure 1. The intracellular delivery routes of different drug formulations. Chemotherapeutics was easily pumped out by P-gp overexpressed on MDR cells (route ①). Chemotherapeutics loaded nanoparticles could enter MDR cells via endocytosis and escapethe P-gp efficiently by “P-gp bypassing effect”, while the released drug can be pumped out again by P-gp located on the cell membrane or on the organelle membrane (route ②). Chemotherapeutics and P-gp inhibitors co-loaded nanoparticles could increase drug accumulations in the cytoplasm and target organelles under the synergy of “P-gp bypassing effect” of nanoparticles and “P-gp inhibition effect” of P-gp inhibitors (route ③).
Figure 2. Characterization of liposomes. Schematic (A) and transmission electron microscopy images (B) of CL-R8-LP (DOX + VER). Release profiles of DOX (C) and VER (D) from different liposomes at 37 °C in 50% FBS, data represent the mean ± SD (n = 3).
Table 1. The size, PDI, Zeta potential and encapsulation efficiency/EE (%) of different liposomes.
Figure 3. (A) P-gp expression in MCF-7 and MCF-7/ADR cells as shown by FACS. Qualitative (B) and quantitative (C) determination of the cellular uptake of different drug formulations by CLSM (Leica, Wetzlar, Germany) and FACS (Beckman Coulter, Fullerton, CA). (a) Blank; (b) DOX; (c) DOX + VER; (d) CL-R8-LP (DOX)/(+Cys); (e) CL-R8-LP (DOX + VER)/(-Cys); (f) CL-R8-LP (DOX + VER)/(+Cys); (g) R8-LP (DOX + VER); (h) CL-LP (DOX + VER)/(+Cys), data represent the mean ± SD (n = 3). *p < 0.001, versus DOX group; $p < 0.001, versus CL-R8-LP (DOX)/(+Cys).
Figure 4. (A) The co-localization of DOX in lysosome after the MCF-7/ADR cells incubating with CL-R8-LP (DOX + VER)/(+Cys) for 0, 2, 4 h, respectively. (B) The endocytosis inhibition assay on MCF-7/ADR cells. The inhibition rate (%) is expressed as the ratios of the cellular uptake in the presence of various inhibitors to the uptake in the absence of inhibitor. Data represent the mean ± SD (n = 3). *p < 0.001, N.S.: No significant difference, versus control group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
Table 2. The IC50, the corresponding resistance index (RI) and Reversal efficiencies (RE) of different DOX formulations in MCF-7 and MCF-7/ADR cells.
Figure 5. The apoptosis assay on MCF-7/ADR cells after treatment with different drug formulations. (A) The representative quadrant plot obtained by FACS analysis showing the effect of drug formulations on initiation of apoptotic activity in vitro. (B) The proportion of apoptotic and necrotic MCF-7/ADR cell death (%) after different DOX formulation treatment. (a) Blank; (b) DOX; (c) DOX + VER; (d) CL-R8-LP (DOX)/(+Cys); (e) CL-R8-LP (DOX + VER)/(-Cys); (f) CL-R8-LP (DOX + VER)/(+Cys); (g) R8-LP (DOX + VER); (h) CL-LP (DOX + VER)/(+Cys). The concentration of DOX (free or equivalent) in the cell culture was 20 μM. Data represent the mean ± SD (n = 3). *p < 0.001, N.S.: No significant difference, versus CL-R8-LP (DOX + VER)/(+Cys) group.
Figure 6. (A) Representative CLSM images of MCF-7 and MCF-7/ADR tumor spheroids incubated with different drug formulations at 37 °C for 4 h. (B) Inhibition ratio (%) of tumor spheroids 7 days after treatment with different drug formulations at 37 °C. (a) Blank; (b) DOX; (c) DOX + VER; (d) CL-R8-LP (DOX)/(+Cys); (e) CL-R8-LP (DOX + VER)/(-Cys); (f) CL-R8-LP (DOX + VER)/(+Cys); (g) R8-LP (DOX + VER); (h) CL-LP (DOX + VER)/(+Cys), data represent the mean ± SD (n = 3). *p < 0.001, N.S.: No significant difference, versus corresponding CL-R8-LP (DOX + VER)/(+Cys) group.
Figure 7. The anti-tumor assay of different DOX formulations in MCF-7/ADR tumor bearing mice. (a) saline; (b) DOX + VER; (c) CL-R8-LP (DOX)/(+Cys); (d) CL-R8-LP (DOX + VER)/(-Cys); (e) CL-R8-LP (DOX + VER)/(+Cys); (f) R8-LP (DOX + VER); (g) CL-LP (DOX + VER)/(+Cys). (A) Tumor growth curves of mice and (B) body weight changes of mice receiving different DOX formulations. The drug administration started on Day 0 and repeated on Day 4, 8, 12 and 16 for a total of five times (indicated as red arrow), while Cys or PBS administration started on Day 1 and repeated on Day 5, 9, 13 and 17 (pointed by the green arrows). (C) Photographs of tumors harvested from each treatment group at the end of treatment. (D) The tumor weight of the excised tumor. (E) Representative images of H&E stained tumor tissues after treatment, the necrosis areas were delineated with the dotted lines. Data represent mean ± SD (n = 6). *p < 0.01, **p < 0.001 versus CL-R8-LP (DOX + VER)/(+Cys) group.
Figure 8. Histological analysis for different organs of MCF-7/ADR tumor bearing mice after treatment. (All tissues: 200×). The analysis showed that DOX + VER resulted in heart toxicity as indicated by the appearance of hyperemia, myocardial fiber breakage and necrosis with acute inflammatory cell infiltration (delineated with the dotted line). In contrast, mice administrated with saline, drug loaded or blank liposomes did not exhibit obvious signs of toxicity.
